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Carcinogenesis Advance Access originally published online on June 13, 2006
Carcinogenesis 2006 27(11):2331-2340; doi:10.1093/carcin/bgl083
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

PYK2 mediates anti-apoptotic AKT signaling in response to benzo[a]pyrene diol epoxide in mammary epithelial cells

Andrew D. Burdick1, Irena D. Ivnitski-Steele, Fredine T. Lauer and Scott W. Burchiel*

The University of New Mexico College of Pharmacy Toxicology Program, 1 University of New Mexico Albuquerque, NM, USA

*To whom correspondence should be addressed at: The University of New Mexico College of Pharmacy Toxicology Program, 1 University of New Mexico, Mail Code MSC09 5360, Albuquerque, NM 87131, USA. Tel: +1 505 272 0920; Fax: +1 505 272 6749; Email: sburchiel{at}salud.unm.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (BaP), are known mammary carcinogens in rodents and may be involved in human breast cancer. The carcinogenicity of BaP has been partially attributed to the formation of the BaP diol epoxide (BPDE), which has been shown to stably bind DNA and act as an initiator. BaP is a complete carcinogen, but the mechanisms for tumor promotion are less well characterized. Previous studies have demonstrated that BPDE enhanced anti-apoptotic signaling through Akt; however, mechanisms for Akt activation by BPDE are not well defined. In the current studies, we found that BPDE increased intracellular Ca2+ concentration in the human mammary epithelial cell line MCF-10A. A peak in Ca2+ concentration at 20 min was followed by increased phosphorylation of Pyk2 at Tyr881 and increased total tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). Consistent with activation of the EGFR, Akt and ERK1/2 phosphorylation was detected in MCF-10A cells treated with BPDE. Pharmacological methods to prevent Ca2+ elevation and EGFR activity, and small-interfering RNA against Pyk2, prevented Akt phosphorylation by BPDE, which suggested that Ca2+, Pyk2 and EGFR activation lay upstream of Akt. In addition, we found that BPDE increased p53 activity and apoptosis in MCF-10A; however, transient transfection of constitutively active Akt attenuated both BPDE-dependent apoptosis and p53 activity. In contrast, apoptosis was enhanced by inhibitors of phosphatidyl inositol 3-kinase (PI3-K). This work demonstrates a novel mechanism for Akt activation by BPDE that occurs through increased Ca2+ concentration, and implicates Ca2+, Pyk2, EGFR and Akt as a potential pathway by which BPDE can inhibit apoptosis and act as a promoter of carcinogenesis.

Abbreviations: BaP, benzo[a]pyrene; BPDE, anti-BaP-7,8-diol-9,10-epoxide; BPQ, benzo[a]pyrene quinone; [Ca2+]i, intracellular Ca2+ concentration; DMSO, dimethyl sulfoxide; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK1/2, extracellular-regulated kinase 1,2; HMEC, human mammary epithelial cell; PAH, polycyclic aromatic hydrocarbon; PCR, polymerase chain reaction; PI, propidium iodide; PI3-K, phosphatidylinositol 3-kinase; TBS, Tris-buffered saline


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP), are widespread environmental pollutants that may contribute to human breast cancer. Definitive epidemiological data correlating PAH exposure with higher breast cancer occurrence are not conclusive, probably because of additional environmental and/or genetic factors that may influence susceptibility (for a review, see ref. 1). However, in vivo and in vitro studies strongly support the hypothesis that PAHs play a causative role in breast cancer (2–6). BaP is a procarcinogen requiring metabolic conversion into more reactive metabolites by cytochrome P450-, peroxidase- and/or microsomal epoxide hydrolase-catalyzed reactions (7). The ultimate carcinogenic metabolite of BaP is often cited as being anti-7,8-dihydrodiol-9,10-epoxy-benzo[a]pyrene (BPDE) owing to the ability of this compound to form stable adducts with DNA and act as an initiator of carcinogenesis (8). It has been well established in vitro that BPDE causes DNA damage and adduct formation, a loss of cell cycle progression and the induction of apoptosis (9–12). However, in vivo, cells that become malignant must replicate damaged DNA, suggesting that they are able to escape cell cycle regulation and/or apoptosis. In fact, some cell types such as fibroblasts (13) and human mammary epithelial cells (HMECs) (14) can show resistance to BPDE-induced apoptosis, which may increase the probability that damaged DNA is replicated.

Our laboratory is interested in identifying the signaling pathways that regulate cell proliferation and apoptosis in mammary cells exposed to PAHs, providing insight into how these compounds promote carcinogenesis. BPDE has been shown to increase the phosphorylation of numerous protein kinases including p38, extracellular-regulated kinase (ERK), Akt and c-jun N-terminal kinase (JNK). Pharmacological and genetic approaches to block p38 have demonstrated that this pathway is at least partially required for the induction of apoptosis by BPDE (10). In addition, a role for phosphatidyl inositol 3-kinase (PI3-K)/Akt in regulating JNK and AP-1 activity has also been demonstrated in vitro (15). The inhibition of apoptosis is well known to be an important aspect of tumor promotion, and the Akt pathway may be highly involved in this process. Increasing evidence indicates that BPDE is not solely an initiator, but can also act as a promoter by simultaneously regulating anti-apoptotic signaling.

We have shown previously that BaP increased intracellular Ca2+ concentration ([Ca2+]i) in human and murine lymphocytes (16–18) and HMECs (2,19). These studies determined that metabolism of BaP is critical for the Ca2+ effect, with BPDE and BaP-quinones (BPQs) being the most important terminal metabolites. Interestingly, BaP metabolites produce differential signaling effects, probably due to their unique structures and ability to interact with other signaling pathways. For example, rapidly increased [Ca2+]i by the novel ortho-BPQ (7,8-BPQ) is dependent upon an interaction with the ryanodine receptor (RyR) (17,20). In contrast, immediate Ca2+ elevation by BPDE in small airway epithelial (SAE) cells is RyR-independent, but sensitive to inositol 1,4,5-triphosphate receptor (InsP3R) inhibitors (21) and could involve PLC{gamma}-dependent mobilization of Ca2+ from InsP3-sensitive stores (16). BPDE and BPQs were also found to cause a more sustained, and later onset of Ca2+ release, which is most probably due to oxidative stress, energy depletion in the mitochondria, followed by a loss in Ca2+ buffering capacity (22).

In vitro cultures of primary HMECs and MCF-10A cells are highly growth factor-dependent, with cells undergoing apoptosis 2–3 days following epidermal growth factor (EGF) and insulin withdrawal (23). However, HMEC survival is enhanced in the absence of growth factors when cells are cultured in the presence of BaP (2). We have partially attributed this survival effect to the production of BPQs because these compounds were shown to activate the EGF receptor (EGFR) through redox-cycling and ROS production (3). However, established tumor promoters such as the phorbol esters and thapsigargin are known to increase intracellular Ca2+ concentration ([Ca2+]i), and growth factor receptors are activated in response to changes in Ca2+ homeostasis in mammary epithelial cells (24). Thus, we hypothesized that BPDE could activate growth factor receptor pathways through a similar mechanism involving increased [Ca2+]i. In this report, we have observed a novel mechanism for Akt activation by BPDE that required increased [Ca2+]i, Pyk2 and EGFR activity. Furthermore, we demonstrate that the activation of Akt by BPDE is functionally significant, resulting in an anti-apoptotic signal that may oppose DNA damage-induced apoptotic signaling. We hypothesize that Akt activation by BPDE may represent a mechanism for tumor promotion by this known mutagenic metabolite of BaP.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials
All chemicals were purchased from Sigma (St Louis, MO, USA), unless otherwise indicated. BPDE was purchased from Midwest Research Institute (Kansas City, MO, USA) at >99% purity and maintained as a stock solution (10 mM) in anhydrous tissue culture grade dimethyl sulfoxide (DMSO) sealed under nitrogen at –20°C. Preparation of PAHs and cell culture treatments were carried out under low ambient lighting. LY294002, PD098059 and SB203580 were purchased from Calbiochem (La Jolla, CA, USA) and stored at –20°C in DMSO. The final concentration of DMSO in all experiments was 0.1%.

MCF-10A cell culture and transfections
MCF-10A cells are a spontaneously immortalized and growth factor-dependent mammary epithelial cell line that is grown on Vitrogen-coated (Collagen Corp., Palo Alto, CA, USA) 100 x 20 mm dishes (Corning Glass, Corning, NY, USA) in serum-free, growth factor-defined media at 10% CO2 and 37°C, as described elsewhere (23). For transient transfections, MCF-10A cells were plated into 6-well culture dishes at 2 x 105 cells per well and chemically transfected with FuGENE® transfection reagent (Roche) per the supplied protocol. The p53-luciferase reporter (0.5 µg per well) was purchased from Stratagene, and the mutant Akt1 constructs (0.1–0.5 µg per well) were purchased from Upstate Biotechnology. Luciferase reporter assays were carried out on a Turner 20/20 Luminometer (Turner Biosystems, Sunnyvale, CA, USA) with Promega's Dual Luciferase assay reagents, and reporter activity was normalized to Ranilla (pRL-TK, Promega) and total cellular protein. For Pyk2 knockdown experiments, MCF-10A cells were transfected with SMARTpool siRNA (#M-003165, Dharmacon, Lafayette, CO, USA) using the manufacturer's guidelines for cell line transfection. Cells (3 x 105 cells/well) were seeded in triplicate on 6-well collagen-coated plates (BD BioCoat) in 2 ml of antibiotic-free growth medium. Following a 24 h incubation period, cells were treated with 100 nM of Pyk2 siRNA or siGLO RNA (negative control) using Dharmafect® transfection agent (4 µl/well). Cells were incubated for an additional 24 h, and then treated as indicated in serum-free media without EGF or insulin.

Measurement of intracellular calcium
MCF-10A cells were cultured on 6-well plates and treated in triplicate as indicated in the figure legends. After treatment, cells were harvested with trypsin, washed once and suspended in 100 µl media containing 2 µM Fluo-3-AM (Molecular Probes) (25) and incubated at 37°C for 1 h. Following the dye incubation, complete media was added to each sample to bring the total volume to 500 µl. Sample order was randomized, and fluorescence intensities were determined by flow cytometry. Mean channel fluorescence was calculated by CellQuest software in at least 10 000 gated cells.

Western blotting
Cells were cultured as above in complete growth medium, and at ~85% confluence, cultured overnight in media without EGF or insulin to reduce basal kinase phosphorylation. Cells were treated as indicated in media without EGF or insulin, and following treatment, washed twice with ice-cold phosphate-buffered saline and collected into cold lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1 µg/ml Leupeptin and 1 mM PMSF). Insoluble material was removed by centrifugation at 16 000x g for 10 min at 4°C and protein concentration was determined (Pierce, Micro-BCA). Equal amounts of protein (10 µg minigels, 50 µg large gels) were loaded onto 10% SDS–PAGE gels, and transferred onto PVDF (NEN Polyscreen®). Equal protein loading and transfer quality was verified by Ponceau-S staining. Membranes were destained with H2O, and blocked with 5% non-fat dry milk (Blocking Grade, Bio-Rad) in Tris-buffered saline (TBS)/0.1% Tween-20 for 1 h at room temperature. Membranes were incubated overnight at 4°C with primary antibodies diluted 1 : 1000 in 5% BSA in TBS/0.1% Tween-20. Detection was carried out the following day by chemiluminescence (NEN Renaissance® Chemiluminescent Reagent) using a horse radish peroxidase-conjugated secondary antibody diluted in 5% non-fat dry milk in TBS/0.1% Tween-20 (1 : 5000, Promega). Alternatively (where noted), membranes were incubated with biotinylated secondary antibodies (1 : 20 000, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature, followed by 0.5 µCi of 125I-labeled streptavidin (3.7 MBq/ml specific activity, GE Healthcare) in TBS/0.1% Tween-20. Rabbit polyclonal antibodies against Pyk2, p-Hdm2, Akt, ERK1/2 and p38 were obtained from Cell Signaling Technologies (Beverly, MA, USA); the p-Pyk2 (Tyr881) antibody and the Mdm2 antibodies were obtained from Biosource (Camarillo, CA, USA); and the anti-actin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). For chemiluminescence, band densities were determined by high-resolution scanning of exposed films followed by analysis with ImageJ software (NIH, Bethesda, MD, USA). For radioisotope experiments, membranes were exposed to phosphorimager screens and analyzed by filmless autoradiography analysis and software (Packard Instruments Cyclone, Optiquant v3.10).

Detection of apoptosis and cell number
Apoptosis was determined by flow cytometry using Annexin-V conjugated to FITC (PharMingen, San Diego, CA, USA) as described previously (23). One of the early changes occurring during apoptosis is a change in the plasma membrane whereby phosphatidylserine (PS) is transposed from the inner to the outer surface (26). Annexin-V is a 35–36 kDa Ca2+-dependent phospholipid binding protein that has a high affinity for PS. Cells are not fixed during staining, allowing for co-staining with propidium iodide (PI), which cannot penetrate intact cellular membranes. To distinguish between apoptosis and necrosis, cells that stained for PI or PI and Annexin-V were determined to be necrotic and not counted as apoptotic. Cell number was determined with the CellTiter 96® Cell Proliferation Assay (Promega) according to supplied instructions. The assay is based on the bioreduction of the tetrazolium reagent (MTS) to a colored formazan product. The absorbance of the product at 490 nm has been reported to be directly proportional to the number of viable cells in culture (27).

Real-time quantitative polymerase chain reaction (PCR)
Pyk2 mRNA induction was measured by real-time quantitative reverse transcriptase PCR. MCF-10A cells were treated in triplicate and MCF-10A RNA was collected using standard techniques as described previously (3). Forward and reverse primers were purchased from Qiagen in a pre-validated QuantiTect SYBR® PCR assay, and PCR reactions were carried out on a Bio-Rad iCycler®.

Statistical analysis
Data were analyzed for statistical differences between control and treated groups using SigmaStat software (Jandel Scientific, San Rafael, CA, USA). ANOVA followed by Dunnett's post tests were performed on sample means.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
BPDE increases intracellular Ca2+ concentration ([Ca2+]i) in MCF-10A
In this study, we hypothesized that rapidly increased [Ca2+]i by BPDE activated growth regulatory pathways in MCF-10A cells. To determine if BPDE elevated intracellular Ca2+, the fluorescent probe Fluo-3-AM was utilized with analysis by flow cytometry (28). With this method, we found that 1 µM BPDE increased Fluo-3 mean channel fluorescence at the earliest time point investigated, that is, 20 min (Figure 1A). A late rise in Ca2+ concentration by BPDE was also observed in MCF-10A cells at 21 h, confirming our previous observations in B-lymphocytes (18). Increased mean channel fluorescence at 20 min and 21 h was significantly different from DMSO controls (P < 0.05) and was accompanied by an obvious shift in population frequencies (Figure 1B, 20 min and 21 h). No effect was observed at intermediate times, indicating that there were two separate peaks in Ca2+ elevation. Our data are consistent with previously published results in SAE cells obtained using single cell ratiometric imaging (21), and provide new evidence demonstrating that BPDE rapidly increased [Ca2+]i in MCF-10A.


Figure 1
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  		Fig. 1 Effect of BPDE on intracellular Ca2+. (A) MCF-10A cells were cultured in triplicate on 6-well culture plates and allowed to reach ~80% confluence before treatment. On the day of the assay, culture media was removed and replaced with warmed media containing 0.1% DMSO or 1 µM BPDE. Following treatment, cells were loaded with 2 µM Fluo-3-AM as described in Materials and methods. For the 20 min treatment time, the cells were loaded with Fluo-3-AM before treatment. Bars represent the mean channel fluorescence ± SEM for 10 000 gated events. Figure represents a single experiment that was repeated with similar results on one other occasion. (B) Histograms of representative samples from Part A. Asterisk represents significantly different from control (P < 0.05).

 
BPDE induces Pyk2, EGFR, Akt, ERK and p38 phosphorylation
After verifying that BPDE elevated Ca2+ in MCF-10A, we next sought to determine if there was enhanced Ca2+-related signaling, focusing specifically on events subsequent to the first rise in [Ca2+]i. To this end, we measured the phosphorylation of Pyk2, a Ca2+-dependent protein tyrosine kinase. To quantify immunoreactivity, 125I-labeled streptavidin was utilized with analysis carried out as described in Materials and methods. In MCF-10A, BPDE increased the phosphorylation of Pyk2 at Tyr881, which peaked at 40 min and then diminished (Figure 2A, P-Pyk2). Phosphorylation of Pyk2 at Tyr881 is known to link Pyk2 with growth factor receptor-bound protein 2 (Grb2), leading to the activation of ERK1/2 (29). Additionally, Pyk2 has been associated with the activation of the EGFR (30,31) and PDK1 (32). Therefore, we also determined the phosphorylation states of the EGFR, Akt and ERK1/2, because these signaling molecules represented downstream effectors that could have been activated by Pyk2. In the identical cell lysates tested for Pyk2 phosphorylation, we found increased tyrosine phosphorylation of the 180 kd EGFR between 1 and 3 h and increased Akt and ERK1/2 phosphorylation that peaked between 3 and 4 h (Figure 2A). Because BPDE is known to cause genotoxic stress, we also measured the phosphorylation of the mitogen-activated protein (MAP) kinase p38, as well as Hdm2 (Mdm2, mouse ortholog), the primary regulator of p53. We found that BPDE transiently increased the phosphorylation of p38 and Hdm2, and increased total p53 protein levels after 4 h of BPDE exposure (Figure 2A). Interestingly, the phosphorylation kinetics of p38 and Hdm2 were similar, but differed from those of Akt and ERK1/2. This finding indicated that the Akt/ERK1/2 and p38/Hdm2 pathways were differentially activated. In all experiments, the increased phosphorylated proteins detected were not due to increased total protein because no changes in total protein levels were detected in response to DMSO or BPDE (data not shown).


Figure 2
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  		Fig. 2 Effect of BPDE on Pyk2, Akt, ERK1/2 and p38 phosphorylation. MCF-10A cells were cultured in complete growth media and allowed to reach ~80% confluence. Growth media was removed and replaced overnight with serum-free media without EGF or insulin. Cells were treated the following day with fresh media containing 0.1% DMSO or 1 µM BPDE, and protein phosphorylation was determined by western blotting as described in the text. (A) The time course of kinase phosphorylation was determined from 20 min to 4 h following BPDE treatment. Vehicle control lanes are displayed only for the first and last treatment time, but the vehicle did not influence kinase phosphorylation at any time point (data not shown). In this part, band densities were determined by quantitative western blotting using 125I-labeled streptavidin. (B) Top panel: MCF-10A cells were pre-treated for 15 min with 0.05% DMSO or 15 µM BAPTA-AM. Next, an additional 0.05% DMSO, 1 µM BPDE or 1 µM A23187 (A2) was added for the time indicated. Cells were harvested and Pyk2 phosphorylation at Tyr881 determined by western blotting. Bottom panel: MCF-10A cells were pre-treated for 15 min with AG1478 or BAPTA-AM at the indicated concentrations before treatment with 1 µM BPDE for 2 h. Akt, ERK1/2 and p38 phosphorylation was determined by western blotting. In the experiment shown, intervening sample lanes between lanes 4 and 7 have been removed; however, the p-Akt, p-ERK1/2 and p-p38 western blots shown in the figure were derived from the same membrane. (C) Western blotting was conducted following treatment with 0.1% DMSO, 1 µM BPDE (B), 1 µM Thapsigargin (Tg), 1 µM ionomycin (Io) or 1 µM A23187 (A2) for 2 h. Where indicated, cells were pre-treated for 15 min with 1 µM AG1478. As a positive control, MCF-10A cells were treated for 15 min with 10 ng/ml EGF (E) with or without AG1478. Western blotting was conducted with phospho- or pan-specific antibodies against Akt or ERK1/2. The results in all parts were repeated with identical results in multiple experiments.

 
Because the kinetics of Akt and ERK1/2 phosphorylation trailed that of Pyk2 and EGFR, we believed that the early rise in intracellular Ca2+ initiated this particular signaling cascade. To test this hypothesis, we determined if the intracellular Ca2+ chelator BAPTA-AM prevented Pyk2, Akt and ERK1/2 phosphorylation by BPDE. BAPTA-AM blocked Pyk2 phosphorylation at 30 min by BPDE and by the positive control A23187 [GenBank] (A2) (Figure 2B, top panel). In addition, Akt and ERK1/2 phosphorylation was attenuated by BAPTA-AM and the EGFR inhibitor, AG1478 (Figure 2B, bottom panels). Interestingly, Akt phosphorylation was more sensitive to inhibition by BAPTA-AM than ERK1/2. Although ERK1/2 phosphorylation was slightly attenuated, BAPTA-AM alone could not lower ERK1/2 phosphorylation back to basal levels, indicating an additional signaling component contributed to ERK activation. Neither the Ca2+ chelator nor the EGFR inhibitor reduced p38 phosphorylation; in fact, slightly exacerbated p38 phosphorylation was observed by both inhibitors. This suggested that the activation of p38 occurred through a separate mechanism that was not dependent upon Ca2+ elevation or EGFR activity, and is consistent with the altered time course of p38 phosphorylation that was detected in Figure 2A.

Next, we tested whether compounds known to mimic and/or increase [Ca2+]i could similarly enhance Akt and ERK1/2 phosphorylation in MCF-10A cells. The sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor thapsigargin (Tg), as well as the Ca2+ ionophores ionomycin (Io) and A23187 [GenBank] (A2), each increased Akt and ERK1/2 phosphorylation, and this activity was similarly inhibited by AG1478 (Figure 2C). As would be expected, we also found that BAPTA-AM inhibited the induction of Akt and ERK1/2 by thapsigargin and the Ca2+ ionophores (data not shown). No changes in the total protein levels of Akt or ERK1/2 were observed by any of the pharmacological agents, confirming that the effects observed are phosphorylation-specific signaling events (Figure 2C).

Pyk2 small-interfering RNA (siRNA) attenuates Akt phosphorylation
To more specifically determine if Pyk2 was involved in the activation of Akt and ERK1/2, an alternative approach using siRNA directed against Pyk2 was utilized. We routinely achieved a reduction in Pyk2 protein of >60% with targeted siRNA, whereas control non-targeted siRNA did not reduce Pyk2 protein expression (Figure 3A). Triplicate wells of MCF-10A cells were pre-treated with control or Pyk2 siRNA, and then subsequently tested for Akt, ERK1/2 and p38 phosphorylation after treatment with BPDE for 2 h. The knockdown of Pyk2 resulted in a significant reduction in Akt phosphorylation, implicating that Pyk2 was an upstream activator of Akt (p-Akt, Figure 3A). In contrast, ERK1/2 and p38 phosphorylation was not significantly reduced by Pyk2 siRNA (p-ERK1/2, p-p38; Figure 3A). This finding confirmed the findings with BAPTA-AM and supported the idea that there was an additional component to the ERK signal contributing to increased phosphorylation and verified that p38 activation occurred through a Pyk2-independent mechanism. Interestingly, elevated total Pyk2 protein was observed in BPDE-treated control siRNA cells at 2 h (Pyk2; Figure 3A). However, experiments using quantitative PCR did not show an increase in Pyk2 mRNA by BPDE alone (Figure 3B), and no significant differences were noted in total Pyk2 protein levels in BPDE-treated cells (Figure 2A). Therefore, this effect appeared to be a secondary effect of the control siRNA.


Figure 3
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  		Fig. 3 Pyk2 mediates Akt phosphorylation by BPDE. (A) MCF-10A cells were transfected in triplicate with siRNA against Pyk2 or non-targeted control siRNA 24 h before treatment. Samples were treated with DMSO or 1 µM BPDE for 2 h, and proteins collected for western blotting as described in the text. The experiment shown represents a single membrane that was initially probed with antibodies against Pyk2, phospho-Akt and phospho-ERK1/2, and following detection, stripped and reprobed with pan-ERK and pan-Akt antibodies. Values with different superscripts are significantly different from each other (P < 0.05). The experiment shown was repeated with similar results in a separate independent experiment. (B) Pyk2 mRNA expression was tested in MCF-10A cells treated 2 or 4 h with 1 µM BPDE using real-time quantitative PCR.

 
Akt attenuates BPDE-induced apoptosis
Our data to this point indicated that Akt phosphorylation was induced by BPDE-dependent Ca2+ elevation, with a concomitant increase in Pyk2 and EGFR activity. Although we detected ERK1/2 and p38 phosphorylation by BPDE, we were specifically interested in determining the functional significance of Akt activity following BPDE exposure in MCF-10A. Because of the known anti-apoptotic role of Akt, we considered the possibility that the activation of Akt was contributing to a pro-survival signal in MCF-10A cells treated with BPDE. Therefore, we examined whether modulation of the Akt pathway would affect BPDE-induced apoptosis in MCF-10A.

Apoptosis following BPDE exposure was determined with Annexin-V conjugated to FITC and analysis by flow cytometry. Co-staining with PI allowed dead and/or necrotic cells to be removed from the measurement of apoptosis because live cells with intact membranes can exclude PI. With this method, a significant percentage of the MCF-10A cell population was classified as early apoptotic (Annexin-V positive, PI negative) beginning at 18 h (Figure 4A). The percentage shown represented a snapshot of the number of early apoptotic cells at the time of the assay, but did not allow for a measurement of the total number of cells that had already undergone apoptosis. When cell number was measured, a much more significant reduction in total cell number was observed (Figure 4B). The flow cytometry analysis suggested that the majority of the cells had died through apoptosis, because only 1.5 ± 0.2% necrotic cells (Annexin-V negative, PI positive) were detected at 24 h (data not shown) versus 4.2 ± 0.3% apoptotic cells.


Figure 4
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  		Fig. 4 BPDE induces apoptosis in MCF-10A cells. (A) MCF-10A cells were cultured in 6-well plates and treated in triplicate with 0.1% DMSO or 1 µM BPDE for the time indicated. Following treatment, attached cells as well as any cells that had detached during the course of the treatment were collected and stained with Annexin V-FITC and PI for 30 min for analysis by flow cytometry. Treatment samples were randomized and the cytometer settings were kept constant throughout the experiment. Data are expressed as the mean percentage of cells that fell into the lower right quadrant of the dotplots (Annexin-V positive/PI negative) ± SEM. One representative experiment that was reproduced on several occasions is shown. (B) MCF-10A cells were seeded into a 96-well plate at 5000 cells per well (n = 6) and treated with 0.1% DMSO or 1 µM BPDE the following day for the time indicated. At each interval, cells were incubated with the reagent for 3 h at 37°C, followed by measurement of absorbance at 490 nm. The absorbance values are shown as a percentage of DMSO controls (C). Asterisk indicates that absorbance values were significantly different from those of controls (P < 0.05), **P < 0.01.

 
Next, we examined the effect of pharmacological inhibitors to Akt, ERK1/2 and p38 on BPDE-induced apoptosis. Co-treatment of cells with BPDE and the PI3-K inhibitor LY294002 increased the percentage of cells undergoing apoptosis at 24 h. In contrast, the MEK1 inhibitor PD98059 had no effect, and the p38 inhibitor SB203580 reduced the percentage of apoptotic cells (Figure 5B). The inhibitors used in this study blocked BPDE-induced phosphorylation of their respective kinases, with the exception of SB203580 because it acts directly on the p38 catalytic site (Figure 5A). Interestingly, Hdm2 phosphorylation at the Akt phosphorylation site (Ser166) was slightly attenuated by LY294002, but completely ablated by SB203580 (Figure 5A). This verified that Hdm2 was a target of Akt in this model system, but also suggested a role for p38 in the regulation of Hdm2 phosphorylation. Importantly, no inhibitor changed the basal level of apoptosis when given alone (Figure 5B, white bars), nor did they reduce cell viability at the concentrations and length of treatment used (data not shown). To verify the effectiveness and specificity of the kinase inhibitors used in these studies, we pre-treated MCF-10A cells for 18 h with 1 µM AG1478, 30 µM LY294002 or 30 µM PD98059 and measured kinase phosphorylation following a 15 min treatment with 10 ng/ml EGF (Figure 5A, bottom panel). As shown, each inhibitor maintained the ability to specifically block the intended pathway.


Figure 5
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  		Fig. 5 Effect of kinase inhibitors on apoptosis. (A) MCF-10A cells were seeded onto 100 mm tissue culture dishes and allowed to reach ~80% confluence. Top panel: Cells were cultured in the absence of insulin and EGF for 18 h to reduce basal Akt and ERK1/2 phosphorylation, and then treated as indicated with vehicle or 1 µM BPDE for 2 h. Where indicated, cells were pre-treated with LY294002 (LY), PD98059 (PD) or SB203580 (SB) for 15 min before BPDE was added. Bottom panel: Cells were cultured as above and kinase inhibitors were added 18 h before treating for 15 min with 10 ng/ml EGF. Protein lysates were collected, and kinase phosphorylation determined by western blotting as described previously. (B) The percentage of apoptotic cells at 24 h was determined as in Figure 4. Cells were treated with 1 µM BPDE in the presence or absence of the following kinase inhibitors: PI3-K inhibitor (LY, 30 µM LY294002); MEK1 inhibitor (PD, 30 µM PD098059); p38 inhibitor (SB, 10 µM SB203580) (C) MCF-10A cells were seeded onto 6-well plates in triplicate. The effect of the Akt pathway on apoptosis was tested by transiently transfecting constitutively active (myr-Akt1) or dominant negative (dn-Akt1) forms of Akt1 before treating for 24 h with BPDE. The empty vector (pUSE) was utilized as a negative control. Asterisk indicates significantly different from DMSO control (P < 0.05) and hash indicates significantly different from BPDE alone (P < 0.05).

 
The results using LY294002 pointed to the PI3-K/Akt signal induced by BPDE as being anti-apoptotic. To directly test if modulation of the Akt signaling pathway altered apoptosis, we transiently transfected constitutively active (myr-Akt1) and dominant negative (dn-Akt1) forms of Akt1 into MCF-10A cells. As demonstrated in Figure 5C, transient transfection of myr-Akt1 attenuated BPDE-induced apoptosis, whereas dn-Akt1 produced a slight, but non-statistically significant increase in apoptotic cells. The myr-Akt1 construct contains an 11 amino acid c-src myristoylation signal at the N-terminus of Akt, which targets Akt to the membrane, leading to enhanced activity. In contrast, the dn-Akt1 construct contains a single-point mutation (K179M) that inactivates the kinase. Thus, a difference in the cellular localization of the dominant negative may explain why its effect in this assay was modest. Nevertheless, the data demonstrating that endogenous Akt is phosphorylated following BPDE treatment, and the effects of LY294002 and myr-Akt1 on apoptosis, together indicate that this pathway represents an anti-apoptotic signal induced by BPDE.

Collectively, these data suggested that the activation of PI3-K/Akt by BPDE provided an anti-apoptotic signal, consistent with the known function of Akt. We found that the inhibition of p38 activity with SB203580 could partially block BPDE-induced apoptosis, and this result is consistent with previous findings that have pointed to p38 as being an important mediator of apoptosis following BPDE exposure (10). Although ERK is reported to provide both anti-apoptotic (33,34) and apoptotic (35) stimuli in other cell models, we did not observe any effect of PD98059 on apoptosis in MCF-10A cells.

Akt modulates apoptosis and p53 activity in MCF-10A
Numerous substrates for Akt have been reported that include apoptosis-inducing molecules such as the Bcl-2 family member BAD (36), caspase-9 (37) and the Forkhead family of transcription factors (38,39). By these means, Akt directly inhibits apoptosis by phosphorylating and inactivating these substrates. In addition, Akt negatively regulates the p53 pathway through phosphorylation of Hdm2 at Ser166, leading to nuclear entry of Hdm2 and enhanced degradation of p53 through the ubiquitin–proteasome pathway (40). Because we detected reduced Hdm2 phosphorylation in the presence of LY294002, we tested whether modulation of Akt activity affected p53 function in MCF-10A. As expected, we found that BPDE increased p53 phosphorylation at Ser15 and Ser20 (Figure 6A), indicating stabilization and activation of the protein. As shown previously in Figure 2A, total p53 protein levels increased after BPDE treatment (Figure 6A). Consistent with increased p53 protein levels, we detected increased p53-driven luciferase reporter activity at 4 h, which verified that endogenous p53 was activated (Figure 6B). However, when MCF-10A cells were co-transfected with myr-Akt1 and subsequently evaluated for p53-luciferase activity, reduced p53 reporter activity was detected (Figure 6C). In contrast, when the dn-Akt1 was used, enhanced BPDE-induced p53 activity was detected (Figure 6C). These data verified that the activation of Akt by BPDE represents a pro-survival signal that may negatively regulate p53-dependent cell cycle arrest and apoptosis.


Figure 6
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  		Fig. 6 BPDE enhances p53 activity. (A) MCF-10A cells were treated from 20 min to 4 h with vehicle or 1 µM BPDE, and proteins were collected in lysis buffer and resolved by SDS–PAGE. PVDF membranes were probed with antibodies against phosphorylated p53 (Ser15 and Ser20) or pan-p53. Antibodies against actin were used to verify equal protein loading between lanes. (B) MCF-10A cells were seeded onto 12-well plates, and sub-confluent cells were transiently transfected with 0.5 µg p53-luciferase and 0.25 µg pRL-TK per well. Cells were allowed to recover overnight, and normalized luciferase activity was subsequently determined following treatment with DMSO or 1 µM BPDE. Data are expressed as fold luciferase control vector, pTA-luc (grey bar). (C) As in Part B, MCF-10A cells were transiently transfected with 0.5 µg pp53-luc and 0.25 µg pRL-TK along with 0.1 µg per well of pUSE, dn-Akt1 or myr-Akt1. Cells were allowed to recover by 24 h, and then treated for 4 h with vehicle or 1 µM BPDE. Each part was verified on at least one additional occasion with a replicate experiment. Asterisk indicates significantly different from control (P < 0.05). Hash indicates significantly different from treatment without Akt1 co-transfection.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
BaP is known as a complete carcinogen, capable of initiation and promotion. Initiation is partly due to metabolism of BaP into mutagenic metabolites, such as BPDE. BPDE–DNA adducts have been demonstrated to preferentially form at mutational hotspots in the p53 tumor suppressor gene (41), initiate a G to T transversion that activates ras (42) and have been detected in human breast tumors (43). In vitro studies have established that high levels of adduct formation result in cell cycle arrest and increased apoptosis, effects that may be mediated in part through p53. An essential role for p53 in assuring efficient nucleotide excision repair of BPDE adducts has also been demonstrated (12). Interestingly, studies have also shown that BPDE activates separate signaling cascades that may be actively opposing pathways that signal apoptosis. The implications of this finding may be an increased likelihood of an initiated cell escaping growth arrest, and replicating a damaged DNA template. Recently, Li et al. (15) reported that BPDE activated Akt in mouse epidermal C141 cells, and this may have represented a mechanism for AP-1 activation and tumor promotion by BPDE. In this report, we addressed the mechanism underlying Akt activation by BPDE in MCF-10A cells, hypothesizing that this pathway was activated by a rapidly increased [Ca2+]i, leading to enhanced Pyk2 activity and transactivation of the EGFR.

Multiple lines of evidence in the current study supported this hypothesis. First, it was shown that BPDE caused a rapid elevation in [Ca2+]i that correlated with increased Pyk2 phosphorylation (Figure 2A). A second, late peak in Ca2+ concentration was also detected; however, this response was most probably tied to mitochondrial dysfunction and apoptosis (17,18,44). We were particularly interested in the first Ca2+ elevation because we believed this initiated Ca2+-related signaling through Pyk2. Studies suggest that Pyk2 can cross-talk with the EGFR, resulting in downstream kinase signaling (45–47). Consistent with this premise, we found increased EGFR tyrosine phosphorylation and higher Akt and ERK1/2 phosphorylation that trailed Pyk2 activation (Figure 2B). The Ca2+ chelator BAPTA-AM and the EGFR inhibitor AG1478 attenuated Akt and ERK1/2 phosphorylation, but did not reduce p38 phosphorylation (Figure 2B). This result further suggested that Ca2+ influx, resulting in Pyk2 activation, lay upstream of Akt and ERK1/2. In contrast, the activation of p38 occurred through a separate pathway that did not require Pyk2. Thapsigargin, ionomycin and A23187 [GenBank] all mirrored the response of BPDE, which established that alterations in Ca2+ homeostasis affected Akt and ERK1/2 phosphorylation in our model system (Figure 2C). It appeared, however, that the Akt pathway was more sensitive to inhibition by BAPTA-AM than the ERK pathway, which suggested the presence of an additional ERK activator. This observation was confirmed with Pyk2 siRNA that reduced BPDE-induced Akt phosphorylation but did not change ERK1/2 or p38 phosphorylation (Figure 3A).

We found that apoptotic and anti-apoptotic pathways were simultaneously activated by BPDE in MCF-10A cells. Although the overall response of MCF-10A cells to BPDE was increased apoptosis (Figure 4A), approaches to block PI3-K/Akt enhanced the number of apoptotic cells following BPDE treatment, suggesting that this signaling pathway provided a significant anti-apoptotic signal (Figure 5A). In contrast, blocking p38 activity with SB203580 reduced the extent of apoptosis (Figure 5A), which confirmed previous reports that this pathway contributes to an apoptotic signal (10). As mentioned above, Akt activation appeared to arise from Ca2+ elevation, leading to Pyk2 and EGFR activation. In contrast, p38 activation occurred more rapidly than Akt, and was independent of Ca2+, Pyk2 or EGFR (Figure 2A). Previous studies have demonstrated that the p38 pathway was activated by genotoxic stress, and this led to p38-dependent phosphorylation of p53 at Ser15 and Ser20 (48). We detected increased phosphorylation of p53 at Ser15 and Ser20, consistent with the premise that the p38 pathway was linked to p53 activation in MCF-10A cells (Figure 6A). Our data suggested, however, that BPDE-dependent Akt activity partially opposed p53 function. Specifically, we found that BPDE-induced p53 reporter activity was attenuated by constitutively active Akt, whereas p53 reporter activity increased in the presence of the dominant negative Akt (Figure 6C). Other studies have linked Akt to the p53 pathway through its ability to phosphorylate Hdm2 at Ser166 and enhance the degradation of p53 (40). Indeed, we detected increased phosphorylation of Hdm2 (Figure 2A) that was partially attenuated by LY294002 (Figure 5A). Interestingly, the kinetics of Hdm2 phosphorylation more closely resembled that of p38, rather than Akt. Additionally, attenuation of p38 activity with SB203580 completely ablated Hdm2 phosphorylation, suggesting that these pathways are linked. Nevertheless, collectively our results support the idea that Akt regulated p53 activity through Hdm2, but also suggest roles for other regulators of Hdm2.

In summary, our results demonstrate that BPDE activates competing pro- and anti-apoptotic pathways in MCF-10A cells. The significant overall finding of this work established that BPDE activates the PI3-K/Akt pathway through a previously unidentified mechanism that required increased Ca2+, Pyk2 and EGFR activity (Figure 7). We hypothesize that p53 mediates cell cycle arrest and apoptosis in response to DNA damage by BPDE, whereas Ca2+ signaling provides an anti-apoptotic signal through PI3-K/Akt. Therefore, DNA damage by BPDE with a concomitant inhibition of apoptosis through the PI3-K/Akt pathway could represent a mechanism for tumor promotion and increased carcinogenesis by BPDE. Potentially, this anti-apoptotic Akt signaling may increase the likelihood that an initiated cell would escape cell cycle arrest and survive to form a malignancy.


Figure 7
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  		Fig. 7 Proposed mechanism for increased Akt activity and repression of apoptosis. BPDE activates conflicting pro- and anti-apoptotic signals in MCF-10A cells. DNA damage results in the phosphorylation of p53 through an upstream DNA repair mechanism, such as ATR. Phosphorylation of p53 prevents its interaction with Mdm2 (Hdm2, human ortholog), a negative regulator, leading to the induction of p53-responsive genes such as p21, followed by cell cycle arrest and apoptosis. In contrast, BPDE increases intracellular Ca2+, leading to enhanced Pyk2 activity. Pyk2 activates PI3-K/Akt through the EGFR, leading to the attenuation of apoptosis-related signaling.

 


   Footnotes
 
1Present address: The Pennsylvania State University, Center for Molecular Toxicology and Carcinogenesis, University Park, PA, 16802, USA Back


   Acknowledgments
 
The authors wish to thank Dr Jeffrey M. Peters (Penn State University) for western blot quantification, Dr Stephen P. Ethier (University of Michigan) for supplying us with the MCF-10A cell line, Ms Gretchen Ray (University of New Mexico) for maintaining the MCF-10A cell line and Dr Timothy Beischlag (Penn State University) for a helpful review of this manuscript.

Grant Support: NIEHS R01 ES07259 (to S.W.B.) and U.S. Army Medical Research and Materiel Command Grant DAMD17-02-1-0512 (to A.D.B.). This work was sponsored in part by the NM NIEHS Center, P30 ES-012072 with UNM Cancer Research and Treatment Center Facilities.

Conflict of Interest Statement: None declared.


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 Discussion
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Received December 19, 2005; revised April 14, 2006; accepted May 17, 2006.


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